Establishment and maintenance of individual cell fates are fundamental for proper metazoan development. Essential to cell type specification during normal development is the coordinated effort of transcription factors and chromatin modifying enzymes to establish the precise transcriptional profiles through space and time that are necessary to achieve the numerous tissue types of the adult form. During development, the Rb/E2F regulatory complex modulates gene expression to establish the balance between proliferation and differentiation in multiple cell types. However, the exact mechanism(s) by which Rb/E2F controls tissue-specific gene expression remain poorly understood. An expanding body of evidence indicates that Rb/E2F functions by using its ability to recruit various chromatin modifying enzymes to distinct subsets of genes to alter the chromatin landscape, thus impacting gene expression throughout maturation of the organism. The goal of this proposal is to dissect the molecular mechanisms by which Rb/E2F defines gene expression programs during the progression from stem to differentiated cell using the germ line of the nematode Caenorhabditis elegans as a model. The C. elegans germ line offers a particularly well-suited in vivo system for such analyses, as all stages of germ cell development from stem cell to differentiated gamete are simultaneously present within the adult gonad. By applying innovative adaptations to current chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) methodologies, Rb/E2F target genes and their transcriptional state will be identified in distinct subpopulations of the germ line. Given the intimate link between methylation of histone tails with transcriptional activation and repression, histone methylation profiles at Rb/E2F target genes will also be identified. Additionally, the recruitment of histone methyltransferases (HMTs) and demethylases (HDMs) to chromatin, as well as the dependence of such recruitment upon a functional Rb pathway, during germ cel differentiation will be analyzed. Altogether, these analyses will elucidate how Rb/E2F interacts with histone methylation systems to modulate gene expression during cellular differentiation within the germ line. Overall, the proposed studies will provide significant advancements in our understanding of Rb function during cell type specification in a defined biological context.